North American eastern equine encephalitis virus (EEEV), an Alphavirus in the family Togaviridae, is classified in Category B of the NIH Priority Pathogens List, and as a high consequence livestock pathogen by the USDA because it is highly lethal for humans and equines, and because effective vaccines and therapies are lacking. In a mouse model of neurovirulent disease, EEEV infection induces only low levels of IFN-?, at least in part as a consequence of the failure to productively infect myeloid cells and lymphoid tissues; a critical type I IFN evasion strategy. An early and profound block on EEEV infectivity for myeloid-lineage cells occurs at the level of gene expression from the incoming vira genome (and presumably also translation of progeny genomes), suggesting that the efficiency with which this essential first step in replication is completed contributes significantly to cell/tissue tropism. We have recently published in Nature that: i) translation of the EEEV genome occurs with ~1,000-fold greater efficiency in mesenchymal than in myeloid cells dictated by nucleotide sequences in the 3' non-translated region (NTR); ii) myeloid cell-specific microRNA, miR142-3p, has four potential binding sites in this region and is responsible for this translation inhibition; and iii) on the other hand, the same region of the 3' NTR is required for replication in mosquito cells and mosquitoes. Specifically, we propose to identify and determine the effect of mutations in the EEEV genome that restore translation in mammalian myeloid-lineage cells in vitro and in vivo; and similarly characterize the effects of these mutations on mosquito cell replication capability. We anticipate that these studies will allow the identificatio and disablement of mechanisms utilized by EEEV to avoid IFN-? activity and, therefore, generate immunogenic, live-attenuated vaccine candidates that will not infect mosquitoes. Also, we will identify a highly restrictive miRNA binding domain which can be used for design of antiviral therapeutics. Finally, we anticipate that our findings regarding the tropism-determining activity of the EEEV 3' NTR will be recapitulated in other viruses and, thus, the studies will have broad applicability.